Introduction
Processed animal proteins, such as poultry by-product meal (PBM), combine high nutritional quality with a low environmental footprint, aligning with circular bioeconomy principles (Galkanda-Arachchige et al., 2020). Meagre (Argyrosomus regius), a high-value whitefish species native to the Adriatic Sea, has gained renewed interest in Mediterranean aquaculture due to its quality, rapid growth, and adaptability to farming conditions (Kružić et al., 2016). Beyond growth and economic performance, evaluating how alternative protein sources shape the gut microbial ecosystem is essential, since intestinal bacteria and fungi play key roles in digestion, nutrient absorption, and immune homeostasis, and can serve as sensitive early indicators of dietary impact on fish health. This study presents the first combined assessment of the bacterial (16S rRNA) and fungal (ITS2) intestinal communities of meagre fed increasing dietary inclusion levels of PBM as a fishmeal substitute.
Material and methods
Three experimental diets incorporating PBM as a partial fishmeal replacement at two inclusion levels (T1, T2), plus a fishmeal-based control diet (T3/CF), were formulated to meet the nutritional requirements of meagre. The feeding trial was conducted in an open-circuit, outdoor water system at the Laboratory of Aquaculture, Institute of Oceanography and Fisheries (Split, Croatia). A total of 110 meagre (initial mean body weight: 1220 ± 154 g) were acclimated for four weeks on a commercial diet before being randomly distributed into six concrete tanks (600 L; 18 fish per tank, two replicate tanks per treatment). At the conclusion of the trial after one year, digesta content was sampled from the proximal (PI) and distal (DI) intestine of fish from each dietary group for gut microbial community characterization (12 replicates per treatment for each intestinal segment). Fungal (ITS2) and bacterial (16S rRNA V3-V4 region) community composition were characterized in parallel by amplicon sequencing on the Illumina platform. Sequences were denoised into amplicon sequence variants (ASVs) using DADA2 and taxonomically classified against the SILVA 138.2 (16S) and UNITE (ITS2) reference databases. Alpha and beta diversity were calculated for both datasets, and group differences were tested using ANOSIM and PERMANOVA (Bray-Curtis).
Results and discussion
The fungal (ITS2) community was diverse and highly individually variable, dominated by Malasseziomycetes, Dothideomycetes, Sordariomycetes, Eurotiomycetes and Saccharomycetes. Alpha diversity was consistently higher in the proximal than distal intestine, and the fishmeal control showed the lowest fungal diversity and evenness in both segments, while beta diversity and ANOSIM/PERMANOVA confirmed that dietary effects on the mycobiome were strongest distally, with T2 showing an intermediate community structure between T1 and the control.
The bacterial (16S) community was overwhelmingly dominated by Gammaproteobacteria across nearly all samples and diets, indicating a comparatively stable core bacterial assemblage relative to the fungal community. In contrast to the mycobiome, bacterial alpha diversity was highest, not lowest, in the fishmeal control, particularly in the distal intestine. Beta diversity, ANOSIM and PERMANOVA converged on a single, striking pattern: the distal intestinal bacterial community of the control group was markedly and significantly distinct from all other diet-by-segment combinations, whereas proximal communities and both PBM-fed groups (T1, T2) remained largely indistinguishable from one another regardless of gut segment.
Taken together, both microbial kingdoms indicate that PBM inclusion reshapes the meagre intestinal microbial ecosystem primarily in the distal intestine, but with contrasting directionality: PBM diets increased fungal diversity and evenness relative to the control, while the bacterial community of PBM-fed fish appeared more homogeneous than the compositionally distinct, more diverse distal bacterial community observed under the fishmeal-based diet. These complementary bacterial and fungal signatures underscore the value of a dual-kingdom amplicon approach for capturing diet-driven shifts in fish gut microbial ecology, and support poultry by-product meal as a viable, sustainable fishmeal alternative without inducing a uniformly destabilizing effect on the meagre gut microbiota.
Acknowledgment
The authors are thankful to all members of the Laboratory of Aquaculture in Split for their help during the feeding trial and sampling. Support for this study has been provided by the project SustainAqua funded by Next Generation EU-National Recovery and Resilience Plan 2021-2026.
References
Galkanda-Arachchige, H.S.C., Wilson, A.E. and Davis, D.A. (2020). Success of fishmeal replacement through poultry by-product meal in aquaculture feed formulations: a meta-analysis. Rev Aquacult, 12: 1624-1636. https://doi.org/10.1111/raq.12401
Kružić, N., Mustać, B., Župan, I. and Čolak, S. (2016). Meagre (Argyrosomus regius Asso, 1801) aquaculture in Croatia. Croat J. Fish, 74(1): 14-19. https://doi.org/10.1515/cjf-2016-0003